Osteoprotegerin binding proteins

ABSTRACT

A novel polypeptide, osteoprotegerin binding protein, involved in osteolcast maturation has been identified based upon its affinity for osteoprotegerin. Nucleic acid sequences encoding the polypeptide, or a fragment, analog or derivative thereof, vectors and host cells for production, methods of preparing osteoprotegerin binding protein, and binding assays are also described. Compositions and methods for the treatment of bone diseases such as osteoporosis, bone loss due to arthritis or metastasis, hypercalcemia, and Paget&#39;s disease are also provided.

This application is a continuation of U.S. Ser. No. 09/211,315, filedDec. 14, 1998, currently pending, which is a continuation of U.S. Ser.No. 08/880,855, filed Jun. 23, 1997, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 08/842,842, filed Apr. 16, 1997,now U.S. Pat. No. 5,843,678, which is/are hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to polypeptides which are involved inosteoclast differentiation. More particularly, the invention relates toosteoprotegerin binding proteins, nucleic acids encoding the proteins,expression vectors and host cells for production of the proteins, andbinding assays. Compositions and methods for the treatment of bonediseases, such as osteoporosis, bone loss from arthritis, Paget'sdisease, and hypercalcemia, are also described.

BACKGROUND OF THE INVENTION

Living bone tissue exhibits a dynamic equilibrium between deposition andresorption of bone. These processes are mediated primarily by two celltypes: osteoblasts, which secrete molecules that comprise the organicmatrix of bone; and osteoclasts, which promote dissolution of the bonematrix and solubilization of bone salts. In young individuals withgrowing bone, the rate of bone deposition exceeds the rate of boneresorption, while in older individuals the rate of resorption can exceeddeposition. In the latter situation, the increased breakdown of boneleads to reduced bone mass and strength, increased risk of fractures,and slow or incomplete repair of broken bones.

Osteoclasts are large phagocytic mutinucleated cells which are formedfrom hematopoietic precursor cells in the bone marrow. Although thegrowth and formation of mature functional osteoclasts is not wellunderstood, it is thought that osteoclasts mature along themonocyte/macrophage cell lineage in response to exposure to variousgrowth-promoting factors. Early development of bone marrow precursorcells to preosteoclasts are believed to mediated by soluble factors suchas tumor necrosis factor-α (TNF-α), tumor necrosis factorβ (TNF-β),interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6), andleukemia inhibitory factor (LIF). In culture, preosteoclasts are formedin the presence of added macrophage colony stimualting factor (M-CSF).These factors act primarily in early steps of osteoclast development.The involvement of polypeptide factors in terminal stages of osteoclastformation has not been extensively reported. It has been reported,however, that parathyroid hormone stimulates the formation and activityof osteoclasts and that calcitonin has the opposite effect, although toa lesser extent.

Recently, a new polypeptide factor, termed osteoprotegerin (OPG), hasbeen described which negatively regulated formation of osteoclasts invitro and in vivo (see co-owned and co-pending U.S. Ser. No. 08/577,788filed Dec. 22, 1995, Ser. No. 08/706,945 filed Sep. 3, 1996, and Ser.No. 08/771,777, filed Dec. 20, 1996, hereby incorporated by reference;and PCT Application No. WO96/26271). OPG dramatically increased the bonedensity in transgenic mice expressing the OPG polypeptide and reducedthe extent of bone loss when administered to ovariectomized rats. Ananalysis of OPG activity in in vitro osteoclast formation revealed thatOPG does not interfere with the growth and differentiation ofmonocyte/macrophage precursors, but more likely blocks thedifferentiation of osteoclasts from monocyte/macrophage precursors. ThusOPG appears to have specificity in regulating the extent of osteoclastformation.

OPG comprises two polypeptide domains having different structural andfunctional properties. The amino-terminal domain spanning about residues22-194 of the full-length polypeptide (the N-terminal methionine isdesignated residue 1) shows homology to other members of the tumornecrosis factor receptor (TNFR) family, especially TNFR-2, throughconservation of cysteine rich domains characteristic of TNFR familymembers. The carboxy terminal domain spanning residues 194-401 has nosignificant homology to any known sequences. Unlike a number of otherTNFR family members, OPG appears to be exclusively a secreted proteinand does not appear to be synthesized as a membrane associated form.

Based upon its activity as a negative regulator of osteoclast formation,it is postulated that OPG may bind to a polypeptide factor involved inosteoclast differentiation and thereby block one or more terminal stepsleading to formation of a mature osteoclast.

It is therefore an object of the invention to identify polypeptideswhich interact with OPG. Said polypeptides may play a role in osteoclastmaturation and may be useful in the treatment of bone diseases.

SUMMARY OF THE INVENTION

A novel member of the tumor necrosis factor family has been identifiedfrom a murine cDNA library expressed in COS cells screened using arecombinant OPG-Fc fusion protein as an affinity probe. The newpolypeptide is a transmembrane OPG binding protein which is predicted tobe 316 amino acids in length, and has an amino terminal cytoplasmicdomain, a transmembrane doman, and a carboxy terminal extracellulardomain. OPG binding proteins of the invention may be membrane-associatedor may be in soluble form.

The invention provides for nucleic acids encoding an OPG bindingprotein, vectors and host cells expressing the polypeptide, and methodfor producing recombinant OPG binding protein. Antibodies or fragmentsthereof which specifically bind OPG binding protein are also provided.

OPG binding proteins may be used in assays to quantitate OPG levels inbiological samples, identify cells and tissues that display OPG bindingprotein, and identify new OPG and OPG binding protein family members.Methods of identifying compounds which interact with OPG binding proteinare also provided. Such compounds include nucleic acids, peptides,proteins, carbohydrates, lipids or small molecular weight organicmolecules and may act either as agonists or antagonists of OPG bindingprotein activity.

OPG binding proteins are involved in osteoclast differentiation and thelevel of osteoclast activity in turn modulates bone resorption. OPGbinding protein agonists and antagonists modulate osteoclast formationand bone resorption and may be used to treat bone diseases characterizedby changes in bone resorption, such as osteoporosis, hypercalcemia, boneloss due to arthritis metastasis, immobilization or periodontal disease,Paget's disease, osteopetrosis, prosthetic loosening and the like.Pharmaceutical compositions comprising OPG binding proteins and OPGbinding protein agonists and antagonists are also encompassed by theinvention.

DESCRIPTION OF THE FIGURES

FIG. 1. Structure and sequence of the 32D-F3 insert encoding OPG bindingprotein. Predicted transmembrane domain and sites for asparagine-linkedcarbohydrate chains are underlined.

FIG. 2. OPG binding protein expression in COS-7 cells transfected withpcDNA/32D-F3. Cells were lipofected with pcDNA/32D-F3 DNA, the assayedfor binding to either goat anti-human IgG1 alkaline phosphataseconjugate (secondary alone), human OPG[22-201]-Fc plus secondary(OPG-Fc), or a chimeric ATAR extracellular domain-Fc fusion protein(sATAR-Fc). ATAR is a new member of the TNFR superfamily, and thesATAR-Fc fusion protein serves as a control for both human IgG1 Fcdomain binding, and generic TNFR releated protein, binding to 32D cellsurface molecules.

FIG. 3. Expression of OPG binding protein in human tissues. Northernblot analysis of human tissue mRNA (Clontech) using a radiolabeled32D-F3 derived hybridization probe. Relative molecular mass is indicatedat the left in kilobase pairs (kb). Arrowhead on right side indicatesthe migration of an approximately 2.5 kb transcript detected in lymphnode mRNA. A very faint band of the same mass is also detected in fetalliver.

FIG. 4. Structure and sequence of the pcDNA/hu OPGbp 1.1 insert encodingthe human OPG binding protein. The predicted transmembrane domain andsite for asparagine-linked charbohydrate chains are underlined.

FIG. 5. Stimulation of osteoclast development in vitro from bone marrowmacrophage and ST2 cell cocultures treated with recombinant murine OPGbinding protein [158-316]. Cultures were treated with varyingconcentrations of murine OPG binding protein ranging from 1.6 to 500ng/ml. After 8-10 days, cultures were lysed, and TRAP activity wasmeasured by solution assay. In addition, some cultures weresimultaneously treated with 1, 10, 100, 500, and 1000 ng/ml ofrecombinant murine OPG [22-401]-Fc protein. Murine OPG binding proteininduces a dose-dependent stimulation in osteoclast formation, whereasOPG [22-401]-Fc inhibits osteoclast formation.

FIG. 6. Stimulation of osteoclast development from bone marrowprecursors in vitro in the presence of M-CSF and murine OPG bindingprotein [158-316]. Mouse bone marrow was harvested, and cultured in thepresence 250, 500, 1000, and 2000 U/ml of M-CSF. Varying concentrationsof OPG binding protein [158-316], ranging from 1.6 to 500 ng/ml, wereadded to these same cultures. Osteoclast development was measured byTRAP solution assay.

FIG. 7. Osteoclasts derived from bone marrow cells in the presence ofboth M-CSF and OPG binding protein [158-316] resorb bone in vitro. Bonemarrow cells treated with either M-CSF, OPG binding protein, or withboth factors combined, were plated onto bone slices in culture wells,and were allowed to develop into mature osteoclasts. The resultingcultures were then stained with Toluidine Blue (left column), orhistochemically to detect TRAP enzyme activity (right column). Incultures receiving both factors, mature osteoclasts were formed thatwere capable of eroding bone as judged by the presence of blue stainedpits on the bone surface. This correlated with the presence of multiplelarge, multinucleated, TRAP positive cells.

FIG. 8. Graph showing the whole blood ionized calcium (iCa) levels frommice injected with OPG binding protein, 51 hours after the firstinjection, and in mice also receiving concurrent OPG administration. OPGbinding protein significantly and dose dependently increased iCa levels.OPG (1 mg/kg/day) completely blocked the increase in iCa at a dose ofOPG binding protein of 5 ug/day, and partially blocked the increase at adose of OPG binding protein of 25 ug/day. (*), different to vehicletreated control (p<0.05). (#), OPG treated iCa level significantlydifferent to level in mice receiving that dose of OPG binding proteinalone (p<0.05).

FIG. 9. Radiographs of the left femur and tibia in mice treated with 0,5, 25 or 100 ug/day of OPG binding protein for 3.5 days. There is a dosedependent decrease in bone density evident most clearly in the proximaltibial metaphysis of these mice, and that is profound at a dose of 100ug/day.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides for a polypeptide referred to as an OPG bindingprotein, which specficially binds OPG and is involved in osteoclastdifferentiation. A cDNA clone encoding the murine form of thepolypeptide was identified from a library prepared from a mousemyelomonocytic cell line 32-D and transfected into COS cells.Transfectants were screened for their ability to bind to anOPG[22-201]-Fc fusion polypeptide (Example 1). The nucleic acid sequencerevealed that OPG binding protein is a novel member of the TNF familyand is most closely related to AGP-1, a polypeptide previously describedin co-owned and co-pending U.S. Ser. No. 08/660,562, filed Jun. 7, 1996.(A polypeptide identical to AGP-1 and designated TRAIL is described inWiley et al. Immunity 3, 673-682 (1995)). OPG binding protein ispredicted to be a type II transmembrane protein having a cytoplamsicdomain at the amino terminus, a transmembrane domain, and a carboxyterminal extracellular domain (FIG. 1). The amino terminal cytoplasmicdomain spans about residues 1-48, the transmembrane domain spans aboutresidues 49-69 and the extracellular domain spans about residues 70-316as shown in FIG. 1 (SEQ ID NO:______). The membrane-associated proteinspecifically binds OPG (FIG. 2). Thus OPG binding protein and OPG sharemany characteristics of a receptor-ligand pair although it is possiblethat other naturally-occurring receptors for OPG binding protein exist.

A DNA clone encoding human OPG binding protein was isolated from a lymphnode cDNA library. The human sequence (FIG. 4) is homologous to themurine sequence. Purified soluble murine OPG binding protein stimulatedosteoclast formation in vitro and induced hypercalcemia and boneresorption in vivo.

OPG binding protein refers to a polypeptide having an amino acidsequence of mammalian OPG binding protein, or a fragment, analog, orderivative thereof, and having at least the activity of binding OPG. Inpreferred embodiments, OPG binding protein is of murine or human origin.In another embodiment, OPG binding protein is a soluble protein having,in one form, an isolated extracellular domain separate from cytoplasmicand transmembrane domains. OPG binding protein is involved in osteoclastdifferentiation and in the rate and extent of bone resorption, and wasfound to stimulate osteoclast formation and stimulate bone resorption.

Nucleic Acids

The invention provides for isolated nucleic acids encoding OPG bindingproteins. As used herein, the term nucleic acid comprises cDNA, genomicDNA, wholly or partially synthetic DNA, and RNA. The nucleic acids ofthe invention are selected from the group consisting of:

a) the nucleic acids as shown in FIG. 1 (SEQ ID NO: ______) and FIG. 4(SEQ ID NO:______);

b) nucleic acids which hybridize to the polypeptide coding regions ofthe nucleic acids shown in FIG. 1 (SEQ ID NO:______) and FIG. 4 (SEQ IDNO:______) and remain hybridized to the nucleic acids under highstringency conditions; and

c) nucleic acids which are degenerate to the nucleic acids of (a) or(b).

Nucleic acid hybridizations typically involve a multi-step processcomprising a first hybridization step to form nucleic acid duplexes fromsingle strands followed by a second hybridization step carried out undermore stringent conditions to selectively retain nucleic acid duplexeshaving the desired homology. The conditions of the first hybridizationstep are generally not crucial, provided they are not of higherstringency than the second hybridization step. Generally, the secondhybridization is carried out under conditions of high stringency,wherein “high stringency” conditions refers to conditions of temperatureand salt which are about 12-20° C. below the melting temperature (T_(m))of a perfect hybrid of part or all of the complementary strandscorresponding to FIG. 1 (SEQ. ID. NO: ______) and FIG. 4 (SEQ IDNO:______). In one embodiment, “high stringency” conditions refer toconditions of about 65° C. and not more than about 1M Na+. It isunderstood that salt concentration, temperature and/or length ofincubation may be varied in either the first or second hybridizationsteps such that one obtains the hybridizing nucleic acid moleculesaccording to the invention. Conditions for hybridization of nucleicacids and calculations of T_(m) for nucleic acid hybrids are describedin Sambrook et al. Molecular Cloning: A Laboratory Manual Cold SpringHarbor Laboratory Press, New York (1989).

The nucleic acids of the invention may hybridize to part or all of thepolypeptide coding regions of OPG binding protein as shown in FIG. 1(SEQ ID NO: ______) and FIG. 4 (SEQ ID NO: ______); and therefore may betruncations or extensions of the nucleic acid sequences shown therein.Truncated or extended nucleic acids are encompassed by the inventionprovided that they retain at least the property of binding OPG. In oneembodiment, the nucleic acid will encode a polypeptide of at least about10 amino acids. In another embodiment, the nucleic acid will encode apolypeptide of at least about 20 amino acids. In yet another embodiment,the nucleic acid will encode a polypeptide of at least about 50 aminoacids. The hybridizing nucleic acids may also include noncodingsequences located 5′ and/or 3′ to the OPG binding protein codingregions. Noncoding sequences include regulatory regions involved inexpression of OPG binding protein, such as promoters, enhancer regions,translational initiation sites, transcription termination sites and thelike.

In preferred embodiments, the nucleic acids of the invention encodemouse or human OPG binding protein. Nucleic acids may encode a membranebound form of OPG binding protein or soluble forms which lack afunctional transmembrane region. The predicted transmembrane region formurine OPG binding protein includes amino acid residues 49-69 inclusiveas shown in FIG. 1 (SEQ. ID. NO: ______). The predicted transmembraneregion for human OPG binding protein includes residues 49-69 as shown inFIG. 4 (SEQ ID NO:______). Substitutions which replace hydrophobic aminoacid residues in this region with neutral or hydrophilic amino acidresidues would be expected to disrupt membrane association and result insoluble OPG binding protein. In addition, deletions of part or all thetransmembrane region would also be expected to produce soluble forms ofOPG binding protein. Nucleic acids encoding amino acid residues 70-316as shown in FIG. 1 (SEQ ID NO:______), or fragments and analogs thereof,encompass soluble OPG binding proteins.

Nucleic acids encoding truncated forms of soluble human OPG bindingproteins are also included. Soluble forms include residues 69-317 asshown in FIG. 4 (SEQ ID NO: ______) and truncations thereof. In oneembodiment, N-terminal truncations generate polypeptides from residues,70-317, 71-317, 72-317, and so forth. In another embodiment, nucleicacids encode soluble OPGbp comprising residues 69-317 and N-terminaltruncations thereof up to OPGbp [158-317], or alternatively, up to OPGbp[166-317].

Plasmid phuOPGbp 1.1 in E. coli strain DH10 encoding human OPG bindingprotein was deposited with the American Type Culture Collection,Rockville, Md. on Jun. 13, 1997.

Nucleic acid sequences of the invention may be used for the detection ofsequences encoding OPG binding protein in biological samples. Inparticular, the sequences may be used to screen cDNA and genomiclibraries for related OPG binding protein sequences, especially thosefrom other species. The nucleic acids are also useful for modulatinglevels of OPG binding protein by anti-sense technology or in vivo geneexpression. Development of transgenic animals expressing OPG bindingprotein is useful for production of the polypeptide and for the study ofin vivo biological activity.

Vectors and Host Cells

The nucleic acids of the invention will be linked with DNA sequences soas to express biologically active OPG binding protein. Sequencesrequired for expression are known to those skilled in the art andinclude promoters and enhancer sequences for initiation of RNAsynthesis, transcription termination sites, ribosome binding sites forthe initiation of protein synthesis, and leader sequences for secretion.Sequences directing expression and secretion of OPG binding protein maybe homologous, i.e., the sequences are identical or similar to thosesequences in the genome involved in OPG binding protein expression andsecretion, or they may be heterologous. A variety of plasmid vectors areavailable for expressing OPG binding protein in host cells (see, forexample, Methods in Enzymology v. 185, Goeddel, D. V. ed., AcademicPress (1990)). For expression in mammalian host cells, a preferredembodiment is plasmid pDSRα described in PCT Application No. 90/14363.For expression in bacterial host cells, preferred embodiments includeplasmids harboring the lux promoter (see co-owned and co-pending U.S.Ser. No. 08/577,778, filed Dec. 22, 1995). In addition, vectors areavailable for the tissue-specific expression of OPG binding protein intransgenic animals. Retroviral and adenovirus-based gene transfervectors may also be used for the expression of OPG binding protein inhuman cells for in vivo therapy (see PCT Application No. 86/00922).

Procaryotic and eucaryotic host cells expressing OPG binding protein arealso provided by the invention. Host cells include bacterial, yeast,plant, insect or mammalian cells. OPG binding protein may also beproduced in transgenic animals such as mice or goats. Plasmids andvectors containing the nucleic acids of the invention are introducedinto appropriate host cells using transfection or transformationtechniques known to one skilled in the art. Host cells may contain DNAsequences encoding OPG binding protein as shown in FIG. 1 or a portionthereof, such as the extracellular domain or the cytoplasmic domain.Nucleic acids encoding OPG binding proteins may be modified bysubstitution of codons which allow for optimal expression in a givenhost. At least some of the codons may be so-called preference codonswhich do not alter the amino acid sequence and are frequently found ingenes that are highly expressed. However, it is understood that codonalterations to optimize expression are not restricted to theintroduction of preference codons. Examples of preferred mammalian hostcells for OPG binding protein expression include, but are not limited toCOS, CHOd-, 293 and 3T3 cells. A preferred bacterial host cell isEscherichia coli.

Polypeptides

The invention also provides OPG binding protein as the product ofprocaryotic or eucaryotic expression of an exogenous DNA sequence, i.e.,OPG binding protein is recombinant OPG binding protein. Exogenous DNAsequences include cDNA, genomic DNA and synthetic DNA sequences. OPGbinding protein may be the product of bacterial, yeast, plant, insect ormammalian cells expression, or from cell-free translation systems. OPGbinding protein produced in bacterial cells will have an N-terminalmethionine residue. The invention also provides for a process ofproducing OPG binding protein comprising growing procaryotic oreucaryotic host cells transformed or transfected with nucleic acidsencoding OPG binding protein and isolating polypeptide expressionproducts of the nucleic acids.

Polypeptides which are mamalian OPG binding proteins or are fragments,analogs or derivatives thereof are encompassed by the invention. In apreferred embodiment, the OPG binding protein is human OPG bindingprotein. A fragment of OPG binding protein refers to a polypeptidehaving a deletion of one or more amino acids such that the resultingpolypeptide has at least the property of binding OPG. Said fragmentswill have deletions originating from the amino terminal end, the carboxyterminal end, and internal regions of the polypeptide. Fragments of OPGbinding protein are at least about ten amino acids, at least about 20amino acids, or at least about 50 amino acids in length. In preferredembodiments, OPG binding protein will have a deletion of one or moreamino acids from the transmembrane region (amino acid residues 49-69 asshown in FIG. 1), or, alternatively, one or more amino acids from theamino-terminus up to and/or including the transmembrane region (aminoacid residues 1-49 as shown in FIG. 1). In another embodiment, OPGbinding protein is a soluble protein comprising, for example, amino acidresidues 69-316, or 70-316, or N-terminal or C-terminal truncated formsthereof, which retain OPG binding activity. OPG binding protein is alsoa human soluble protein as shown in FIG. 4 comprising residues 69-317 asshown in FIG. 4 and N-terminal truncated forms thereof, e.g., 70-517,71-517, 71-317, 72-317 and so forth. In a preferred embodiment, thesoluble human OPG binding protein comprising residues 69-317 andN-terminal truncation thereof up to OPGbp [158-317], or alternatively upto OPG [166-317].

An analog of an OPG binding protein refers to a polypeptide having asubstitution or addition of one or more amino acids such that theresulting polypeptide has at least the property of binding OPG. Saidanalogs will have substitutions or additions at any place along thepolypeptide. Preferred analogs include those of soluble OPG bindingproteins. Fragments or analogs may be naturally occurring, such as apolypeptide product of an allelic variant or a mRNA splice variant, orthey may be constructed using techniques available to one skilled in theart for manipulating and synthesizing nucleic acids. The polypeptidesmay or may not have an amino terminal methionine residue

Also included in the invention are derivatives of OPG binding proteinwhich are polypeptides that have undergone post-translationalmodifications (e.g., addition of N-linked or O-linked carbohydratechains, processing of N-terminal or C-terminal ends), attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or O-linked carbohydrate chains, and addition of an N-terminalmethionine residue as a result of procaryotic host cell expression. Inparticular, chemically modified derivatives of OPG binding protein whichprovide additional advantages such as increased stability, longercirculating time, or decreased immunogenicity are contemplated. Ofparticular use is modification with water soluble polymers, such aspolyethylene glycol and derivatives thereof (see for example U.S. Pat.No. 4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties. Polypeptides may also be modified at pre-determinedpositions in the polypeptide, such as at the amino terminus, or at aselected lysine or arginine residue within the polypeptide. Otherchemical modificaitons provided include a detectable label, such as anenzymatic, fluorescent, isotopic or affinity label to allow fordetection and isolation of the protein.

OPG binding protein chimeras comprising part or all of an OPG bindingprotein amino acid sequence fused to a heterologous amino acid sequenceare also included. The heterologous sequence may be any sequence whichallows the resulting fusion protein to retain the at least the activityof binding OPG. In a preferred embodiment, the carboxy terminalextracellular domain of OPG binding protein is fused to a heterologoussequence. Such sequences include heterologous cytoplasmic domains thatallow for alternative intracellular signalling events, sequences whichpromote oligomerization such as the Fc region of IgG, enzyme sequenceswhich provide a label for the polypeptide, and sequences which provideaffinity probes, such as an antigen-antibody recognition.

The polypeptides of the invention are isolated and purified from tissuesand cell lines which express OPG binding protein, either extracted fromlysates or from conditioned growth medium, and from transformed hostcells expressing OPG binding protein. OPG binding protein may beobtained from murine myelomonocytic cell line 32-D (ATCC accession no.CRL-11346). Human OPG binding protein, or nucleic acids encoding same,may be isolated from human lymph node or fetal liver tissue. IsolatedOPG binding protein is free from association with human proteins andother cell constituents.

A method for the purification of OPG binding protein from naturalsources (e.g. tissues and cell lines which normally express OPG bindingprotein) and from transfected host cells is also encompassed by theinvention. The purification process may employ one or more standardprotein purification steps in an appropriate order to obtain purifiedprotein. The chromatography steps can include ion exchange, gelfiltration, hydrophobic interaction, reverse phase, chromatofocusing,affinity chromatography employing an anti-OPG binding protein antibodyor biotin-streptavidin affinity complex and the like.

Antibodies

Antibodies specifically binding the polypeptides of the invention arealso encompassed by the invention. The antibodies may be produced byimmunization with full-length OPG binding protein, soluble forms of OPGbinding protein, or a fragment thereof. The antibodies of the inventionmay be polyclonal or monoclonal, or may be recombinant antibodies, suchas chimeric antibodies wherein the murine constant regions on light andheavy chains are replaced by human sequences, or CDR-grafted antibodieswherein only the complementary determining regions are of murine origin.Antibodies of the invention may also be human antibodies prepared, forexample, by immunization of transgenic animals capable of producinghuman antibodies (see, for example, PCT Application No. WO93/12227). Theantibodies are useful for detecting OPG binding protein in biologicalsamples, thereby allowing the identification of cells or tissues whichproduce the protein In addition, antibodies which bind to OPG bindingprotein and block interaction with other binding compounds may havetherapeutic use in modulating osteoclast differentiation and boneresorption.

Antibodies to the OPG binding protein may be useful in treatment of bonediseases such as, osteoporosis and Paget's disease. Antibodies can betested for binding to the OPG binding protein in the absence or presenceof OPG and examined for their ability to inhibit ligand (OPG bindingprotein) mediated osteoclastogenesis and/or bone resorption. It is alsoanticipated that the peptides themselves may act as an antagonist of theligand:receptor interaction and inhibit ligand-mediatedosteoclastogenesis, and peptides of the OPG binding protein will beexplored for this purpose as well.

Compositions

The invention also provides for pharmaceutical compositions comprising atherapeutically effective amount of the OPG binding protein of theinvention together with a pharmaceutically acceptable diluent, carrier,solubilizer, emulsifier, preservative and/or adjuvant. The inventionalso provides for pharmaceutical compositions comprising atherapeutically effective amount of an OPG binding protein agonist orantagonist. The term “therapeutically effective amount” means an amountwhich provides a therapeutic effect for a specified condition and routeof administration. The composition may be in a liquid or lyophilizedform and comprises a diluent (Tris, acetate or phosphate buffers) havingvarious pH values and ionic strengths, solubilizer such as Tween orPolysorbate, carriers such as human serum albumin or gelatin,preservatives such as thimerosal or benzyl alcohol, and antioxidantssuch as ascrobic acid or sodium metabisulfite. Selection of a particularcomposition will depend upon a number of factors, including thecondition being treated, the route of administration and thepharmacokinetic parameters desired. A more extensive survey of componentsuitable for pharmaceutical compositions is found in Remington'sPharmaceutical Sciences, 18th ed. A. R. Gennaro, ed. Mack, Easton, Pa.(1980).

In a preferred embodiment, compositions comprising soluble OPG bindingproteins are also provided. Also encompassed are compositions comprisingsoluble OPG binding protein modified with water soluble polymers toincrease solubility, stability, plasma half-life and bioavailability.Compositions may also comprise incorporation of soluble OPG bindingprotein into liposomes, microemulsions, micelles or vesicles forcontrolled delivery over an extended period of time. Soluble OPG bindingprotein may be formulated into microparticles suitable for pulmonaryadministration.

Compositions of the invention may be administered by injection, eithersubcutaneous, intravenous or intramuscular, or by oral, nasal, pulmonaryor rectal administration. The route of administration eventually chosenwill depend upon a number of factors and may be ascertained by oneskilled in the art.

The invention also provides for pharmaceutical compositions comprising atherapeutically effective amount of the nucleic acids of the inventiontogether with a pharmaceutically acceptable adjuvant. Nucleic acidcompositions will be suitable for the delivery of part or all of thecoding region of OPG binding protein and/or flanking regions to cellsand tissues as part of an anti-sense therapy regimen.

Methods of Use

OPG binding proteins may be used in a variety of assays for detectingOPG and characterizing interactions with OPG. In general, the assaycomprises incubating OPG binding protein with a biological samplecontaining OPG under conditions which permit binding to OPG to OPGbinding protein, and measuring the extent of binding. OPG may bepurified or present in mixtures, such as in body fluids or culturemedium. Assays may be developed which are qualitative or quantitative,with the latter being useful for determining the binding parameters(affinity constants and kinetics) of OPG to OPG binding protein and forquantitating levels of biologically active OPG in mixtures. Assays mayalso be used to evaluate the binding of OPG to fragments, analogs andderivatives of OPG binding protein and to identify new OPG and OPGbinding protein family members.

Binding of OPG to OPG binding protein may be carried out in severalformats, including cell-based binding assays, membrane binding assays,solution-phase assays and immunoassays. In general, trace levels oflabeled OPG are incubated with OPG binding protein samples for aspecified period of time followed by measurement of bound OPG byfiltration, electrochemiluminescent (ECL, ORIGEN system by IGEN),cell-based or immunoassays. Homogeneous assay technologies forradioactivity (SPA; Amersham) and time resolved fluoresence (HTRF,Packard) can also be implemented. Binding is detected by labeling OPG oran anti-OPG antibody with radioactive isotopes (125I, 35S, 3H),fluorescent dyes (fluorescein), lanthanide (Eu3+) chelates or cryptates,orbipyridyl-ruthenium (Ru2+) complexes. It is understood that the choiceof a labeled probe will depend upon the detection system used.Alternatively, OPG may be modified with an unlabled epitope tag (e.g.,biotin, peptides, His₆, myc) and bound to proteins such as streptavidin,anti-peptide or anti-protein antibodies which have a detectable label asdescribed above.

In an alternative method, OPG binding protein may be assayed directlyusing polyclonal or monoclonal antibodies to OPG binding proteins in animmunoassay. Additional forms of OPG binding proteins containing epitopetags as described above may be used in solution and immunoassays.

Methods for indentifying compounds which interact with OPG bindingprotein are also encompassed by the invention. The method comprisesincubating OPG binding protein with a compound under conditions whichpermit binding of the compound to OPG binding protein, and measuring theextent of binding. The compound may be substantially purified or presentin a crude mixture. Binding compounds may be nucleic acids, proteins,peptides, carbohydrates, lipids or small molecular weight organiccompounds. The compounds may be further characterized by their abilityto increase or decrease OPG binding protein activity in order todetermine whether they act as an agonist or an antagonist.

OPG binding proteins are also useful for identification of intracellularproteins which interact with the cytoplasmic domain by a yeasttwo-hybrid screening process. As an example, hybrid constructscomprising DNA encoding the N-terminal 50 amino acids of an OPG bindingprotein fused to a yeast GAL4-DNA binding domain may be used as atwo-hybrid bait plasmid. Positive clones emerging from the screening maybe characterized further to identify interacting proteins. Thisinformation may help elucidate a intracellular signaling mechanismassociated with OPG binding protein and provide intracellular targetsfor new drugs that modulate bone resorption.

OPG binding protein may be used to treat conditions characterized byexcessive bone density. The most common condition is osteopetrosis inwhich a genetic defect results in elevated bone mass and is usuallyfatal in the first few years of life. Osteopetrosis is preferablytreated by administration of soluble OPG binding protein.

The invention also encompasses modulators (agonists and antagonists) ofOPG binding protein and the methods for obtaining them. An OPG bindingprotein modulator may either increase or decrease at least one activityassociated with OPG binding protein, such as ability to bind OPG or someother interacting molecule or to regulate osteoclast maturation.Typically, an agonist or antagonist may be a co-factor, such as aprotein, peptide, carbohydrate, lipid or small molecular weightmolecule, which interacts with OPG binding protein to regulate itsactivity. Potential polypeptide antagonists include antibodies whichreact with either soluble or membrane-associated forms of OPG bindingprotein, and soluble forms of OPG binding protein which comprise part orall of the extracellular domain of OPG binding protein. Molecules whichregulate OPG binding protein expression typically include nucleic acidswhich are complementary to nucleic acids encoding OPG binding proteinand which act as anti-sense regulators of expression.

OPG binding protein is involved in controlling formation of matureosteoclasts, the primary cell type implicated in bone resorption. Anincrease in the rate of bone resorption (over that of bone formation)can lead to various bone disorders collectively referred to asosteopenias, and include osteoporosis, osteomyelitis, hypercalcemia,osteopenia brought on by surgery or steroid administration, Paget'sdisease, osteonecrosis, bone loss due to rheumatoid arthritis,periodontal bone loss, immobilization, prosthetic loosing and osteolyticmetastasis. Conversely, a decrease in the rate of bone resorption canlead to osteopetrosis, a condition marked by excessive bone density.Agonists and antagonists of OPG binding protein modulate osteoclastformation and may be administered to patients suffering from bonedisorders. Agonists and antagonists of OPG binding protein used for thetreatment of osteopenias may be administered alone or in combinationwith a therapeutically effective amount of a bone growth promoting agentincluding bone morphogenic factors designated BMP-1 to BMP-12,transforming growth factor-β and TGF-β family members, fibroblast growthfactors FGF-1 to FGF-10, interleukin-1 inhibitors, TNFα inhibitors,parathyroid hormone, E series prostaglandins, bisphosphonates andbone-enhancing minerals such as fluoride and calcium. Antagonists of OPGbinding proteins may be particularly useful in the treatment ofosteopenia.

The following examples are offered to more fully illustrate theinvention, but are not construed as limiting the scope thereof.

EXAMPLE 1 Identification of a Cell line Source for an OPG BindingProtein

Osteoprotegerin (OPG) negatively regulates osteoclastogenesis in vitroand in vivo. Since OPG is a TNFR-related protein, it is likely tointeract with a TNF-related family member while mediating its effects.With one exception, all known members of the TNF superfamily are type IItransmembrane proteins expressed on the cell surface. To identify asource of an OPG binding protein, recombinant OPG-Fc fusion proteinswere used as immunoprobes to screen for OPG binding proteins located onthe surface of various cell lines and primary hematopoietic cells.

Cell lines that grew as adherent cultures in vitro were treated usingthe following methods: Cells were plated into 24 well tissue cultureplates (Falcon), then allowed to grow to approxiamtely 80% confluency.The growth media was then removed, and the adherent cultures were washedwith phosphate buffered saline (PBS) (Gibco) containing 1% fetal calfserum (FCS). Recombinant mouse OPG [22-194]-Fc and human OPG [22-201]-Fcfusion proteins (see U.S. Ser. No. 08/706,945 filed Sep. 3, 1996) wereindividually diluted to 5 ug/ml in PBS containing 1% FCS, then added tothe cultures and allowed to incubate for 45 min at 0° C. The OPG-Fcfusion protein solution was discarded, and the cells were washed inPBS-FCS solution as described above. The cultures were then exposed tophycoeyrthrin-conguated goat F(ab′) anti-human IgG secondary antibody(Southern Biotechnology Associates Cat. #2043-09) diluted into PBS-FCS.After a 30-45 min incubation at 0° C., the solution was discarded, andthe cultures were washed as described above. The cells were thenanalysed by immunofluorescent microscopy to detect cell lines whichexpress a cell surface OPG binding protein.

Suspension cell cultures were analysed in a similar manner with thefollowing modifications: The diluent and wash buffer consisted ofcalcium- and magnesium-free phosphate buffered saline containing 1% FCS.Cells were harvested from exponentially replicating cultures in growthmedia, pelleted by centrifugation, then resuspended at 1×10⁷ cells/ml ina 96 well microtiter tissue culture plate (Falcon). Cells weresequentially exposed to recombinant OPG-Fc fusion proteins, thensecondary antibody as described above, and the cells were washed bycentrifugation between each step. The cells were then analysed byfluorescence activated cell sorting (FACS) using a Becton DickinsonFACscan.

Using this approach, the murine myelomonocytic cell line 32D (ATCCaccession no. CRL-11346) was found to express a surface molecule whichcould be detected with both the mouse OPG[22-194]-Fc and the humanOPG[22-201]-Fc fusion proteins. Secondary antibody alone did not bind tothe surface of 32D cells nor did purified human IgG1 Fc, indicating thatbinding of the OPG-Fc fusion proteins was due to the OPG moiety. Thisbinding could be competed in a dose dependent manner by the addition ofrecombinant murine or human OPG[22-401] protein. Thus the OPG regionrequired for its biological activity is capable of specifically bindingto a 32D-derived surface molecule.

EXAMPLE 2 Expression Cloning of a Murine OPG Binding Protein

A cDNA library was prepared from 32D mRNA, and ligated into themammalian expression vector pcDNA3.1(+) (Invitrogen, San Diego, Calif.).Exponentially growing 32D cells maintained in the presence ofrecombinant interleukin-3 were harvested, and total cell RNA waspurified by acid guanidinium thiocyanate-phenol-chloroform extraction(Chomczynski and Sacchi. Anal. Biochem. 162, 156-159, (1987)). The poly(A+) mRNA fraction was obtained from the total RNA preparation byadsorption to, and elution from, Dynabeads Oligo (dT) 25 (Dynal Corp)using the manufacturer's recommended procedures. A directional, oligo-dTprimed cDNA library was prepared using the Superscript Plasmid System(Gibco BRL, Gaithersburg, Md.) using the manufacturer's recommendedprocedures. The resulting cDNA was digested to completion with Sal I andNot I restriction endonuclease, then fractionated by size exclusion gelchromatography. The highest molecular weight fractions were selected,and then ligated into the polyliker region of the plasmid vectorpcDNA3.1(+) (Invitrogen, San Diego, Calif.). This vector contains theCMV promotor upstream of multiple cloning site, and directs high levelexpression in eukaryotic cells. The library was then electroporated intocompetent E. coli (ElectroMAX DH10B, Gibco, N.Y.), and titered on LBagar containing 100 ug/ml ampicillin. The library was then arrayed intosegregated pools containing approximately 1000 clones/pool, and 1.0 mlcultures of each pool were grown for 16-20 hr at 37° C. Plasmid DNA fromeach culture was prepared using the Qiagen Qiawell 96 Ultra Plasmid Kit(catalog #16191) following manufacturer's recommended procedures.

Arrayed pools of 32D cDNA expression library were individuallylipofected into COS-7 cultures, then assayed for the acquisition of acell surface OPG binding protein. To do this, COS-7 cells were plated ata density of 1×10⁶ per ml in six-well tissue culture plates (Costar),then cultured overnight in DMEM (Gibco) containing 10% FCS.Approximately 2 μg of plasmid DNA from each pool was diluted into 0.5 mlof serum-free DMEM, then sterilized by centrifugation through a 0.2 μmSpin-x column (Costar). Simultaneously, 10 μl of Lipofectamine (LifeTechnologies Cat #18324-012) was added to a separate tube containing 0.5ml of serum-free DMEM. The DNA and Lipofectamine solutions were mixed,and allowed to incubate at RT for 30 min. The COS-7 cell cultures werethen washed with serum-free DMEM, and the DNA-lipofectamine complexeswere exposed to the cultures for 2-5 hr at 37° C. After this period, themedia was removed, and replaced with DMEM containing 10% FCS. The cellswere then cultured for 48 hr at 37° C.

To detect cultures that express an OPG binding protein, the growth mediawas removed, and the cells were washed with PBS-FCS solution. A 1.0 mlvolume of PBS-FCS containing 5 μg/ml of human OPG[22-201]-Fc fusionprotein was added to each well and incubated at RT for 1 hr. The cellswere washed three times with PBS-FCS solution, and then fixed in PBScontaining 2% paraformaldehyde and 0.2% glutaraldehyde in PBS at RT for5 min. The cultures were washed once with PBS-FCS, then incubated for 1hr at 65° C. while immersed in PBS-FCS solution. The cultures wereallowed to cool, and the PBS-FCS solution was aspirated. The cultureswere then incubated with an alkaline-phosphatase conjugated goatanti-human IgG (Fc specific) antibody (SIGMA Product # A-9544) at Rt for30 min, then washed three-times with 20 mM Tris-Cl (pH 7.6), and 137 mMNaCl. Immune complexes that formed during these steps were detected byassaying for alkaline phosphatase activity using the Fast Red TR/AS-MXSubstrate Kit (Pierce, Cat. #34034) following the manufacturer'srecommended procedures.

Using this approach, a total of approximately 300,000 independent 32DcDNA clones were screened, represented by 300 transfected pools of 1000clones each. A single well was identifed that contained cells whichacquired the ability to be specifically decorated by the OPG-Fc fusionprotein. This pool was subdivided by sequential rounds of sib selection,yeilding a single plasmid clone 32D-F3 (FIG. 1). 32D-F3 plasmid DNA wasthen transfected into COS-7 cells, which were immunostained with eitherFITC-conjugated goat anti-human IgG secondary antibody alone, human OPG(22-201]-Fc fusion protein plus secondary, or with ATAR-Fc fusionprotein (ATAR also known as HVEM; Montgomery et al. Cell 87, 427-436(1996)) (FIG. 2). The secondary antibody alone did not bind toCOS-7/32D-F3 cells, nor did the ATAR-Fc fusion protein. Only the OPG Fcfusion protein bound to the COS-7/32D-F3 cells, indicating that 32D-F3encoded an OPG binding protein displayed on the surface of expressingcells.

EXAMPLE 3 OPG Binding Protein Sequence

The 32D-F3 clone isolated above contained an approximately 2.3 kb cDNAinsert (FIG. 1), which was sequenced in both directions on an AppliedBiosystems 373A automated DNA sequencer using primer-driven Taqdye-terminator reactions (Applied Biosystems) following themanufacturer's recommended procedures. The resulting nucleotide sequenceobtained was compared to the DNA sequence database using the FASTAprogram (GCG, Univeristy of Wisconsin), and analysed for the presence oflong open reading frames (LORF's) using the “Six-way open reading frame”application (Frames) (GCG, Univeristy of Wisconsin). A LORF of 316 aminoacid (aa) residues beginning at methionine was detected in theappropriate orientation, and was preceded by a 5′ untranslated region ofabout 150 bp. The 5′ untranslated region contained an in-frame stopcodon upstream of the predicted start codon. This indicates that thestructure of the 32D-F3 plasmid is consistent with its ability toutilize the CMV promotor region to direct expression of a 316 aa geneproduct in mammalian cells.

The predicted OPG binding protein sequence was then compared to theexisting database of known protein sequences using a modified version ofthe FASTA program (Pearson, Meth. Enzymol. 183, 63-98 (1990)). The aminoacid sequence was also analysed for the presence of specific motifsconserved in all known members of the tumor necrosis factor (TNF)superfamily using the sequence profile method of (Gribskov et al. Proc.Natl. Acad. Sci. USA 83, 4355-4359 (1987)), as modified by Luethy et al.Protein Sci. 3, 139-146 (1994)). There appeared to be significanthomology throughout the OPG binding protein to several members of theTNF superfamily. The mouse OPG binding protein appear to be most closelyrelated to the mouse and human homologs of both TRAIL and CD40 ligand.Further analysis of the OPG binding protein sequence indicated a strongmatch to the TNF superfamily, with a highly significant Z score of19.46.

The OPG binding protein amino acid sequence contains a probablehydrophobic transmembrane domain that begins at a M49 and extends toL69. Based on this configuration relative to the methionine start codon,the OPG binding protein is predicted to be a type II transmembraneprotein, with a short N-terminal intracellular domain, and a longerC-terminal extracellular domain (FIG. 4). This would be similar to allknown TNF family members, with the exception of lymphotoxin alpha(Nagata and Golstein, Science 267, 1449-1456 (1995)).

EXAMPLE 4 Expression of Human OPG Binding Protein mRNA

Multiple human tissue northern blots (Clontech, Palo Alto, Calif.) wereprobed with a ³²P-dCTP labelled 32D-F3 restriction fragment to detectthe size of the human transcript and to determine patterns ofexpression. Northern blots were prehybridized in 5×SSPE, 50% formamide,5×Denhardt's solution, 0.5% SDS, and 100 μg/ml denatured salmon spermDNA for 2-4 hr at 42° C. The blots were then hybridized in 5×SSPE, 50%formamide, 2× Denhardt's solution, 0.1% SDS, 100 μg/ml denatured salmonsperm DNA, and 5 ng/ml labelled probe for 18-24 hr at 42° C. The blotswere then washed in 2×SSC for 10 min at RT, 1×SSC for 10 min at 50° C.,then in 0.5×SSC for 10-15 min.

Using a probe derived from the mouse cDNA and hybridization understringent conditions, a predominant mRNA species with a relativemolecular mass of about 2.5 kb was detected in lymph nodes (FIG. 3). Afaint signal was also detected at the same relative molecular mass infetal liver mRNA. No OPG binding protein transcripts were detected inthe other tissues examined. The data suggest that expression of OPGbinding protein mRNA was extremely restricted in human tissues. The dataalso indicate that the cDNA clone isolated is very close to the size ofthe native transcript, suggesting 32D-F3 is a full length clone.

EXAMPLE 5 Molecular Cloning of the Human OPG Binding Protein

The human homolog of the OPG binding protein is expressed as anapproximately 2.5 kb mRNA in human peripheral lymph nodes and isdetected by hybridization with a mouse cDNA probe under stringenthybdization conditions. DNA encoding human OPG binding protein isobtained by screening a human lymph node cDNA library by eitherrecombinant bacteriphage plaque, or transformed bacterial colony,hybridiziation methods (Sambrook et al. Molecular Cloning: A LaboratoryManual Cold Spring Harbor Press, New York (1989)). To this the phage orplasmid cDNA library are screened using radioactively-labeled probesderived from the murine OPG binding protein clone 32D-F3. The probes areused to screen nitrocellulose filter lifted from a plated library. Thesefilters are prehybridized and then hybridized using conditions specifiedin Example 4, ultimately giving rise to purified clones of the human OPGbinding protein cDNA. Inserts obtained from any human OPG bindingprotein clones would be sequenced and analysed as described in Example3.

A human lymph node poly A+ RNA (Clontech, Inc., Palo Alto, Calif.) wasanalysed for the presence of OPG-bp transcripts as previously in U.S.Ser. No. 08/577,788, filed Dec. 22, 1995. A northern blot of this RNAsample probed under stringent conditions with a 32P-labeled mouse OPG-bpprobe indicated the presence of human OPG-bp transcripts. An oligodT-primed cDNA library was then synthesized from the lymph node mRNAusing the SuperScript kit (GIBCO life Technologies, Gaithersberg, Md.)as described in example 2. The resulting cDNA was size selected, and thehigh molecular fraction ligated to plasmid vector pcDNA 3.1(+)(Invitrogen, San Diego, Calif.). Electrocompetent E. coli DH10 (GIBCOlife Technologies, Gaithersberg, Md.) were transformed, and 1×10⁶ampicillin resistant transformants were screened by colony hybridizationusing a 32P-labeled mouse OPG binding protein probe.

A plasmid clone of putative human OPG binding protein cDNA was isolated,phuOPGbp-1.1, and contained a 2.3 kp insert. The resulting nucleotidesequence of the phuOPGbp-1.1 insert was approximately 80-85% homologousto the mouse OPG binding protein cDNA sequence. Translation of theinsert DNA sequence indicated the presence of a long open reading framepredicted to encode a 317 aa polypeptide (FIG. 4). Comparison of themouse and human OPG-bp polypeptides shows that they are ˜87% identical,indicating that this protein is highly conserved during evolution.

The human OPG binding protein DNA and protein sequences were not presentin Genbank, and there were no homologus EST sequences. As with themurine homolog, the human OPG binding protein shows strong sequencesimilarity to all members of the TNFα superfamily of cytokines.

EXAMPLE 6 Cloning and Bacterial Expression of OPG Binding Protein

PCR amplification employing the primer pairs and templates describedbelow are used to generate various forms of murine OPG binding proteins.One primer of each pair introduces a TAA stop codon and a unique XhoI orSacII site following the carboxy terminus of the gene. The other primerof each pair introduces a unique NdeI site, a N-terminal methionine, andoptimized codons for the amino terminal portion of the gene. PCR andthermocycling is performed using standard recombinant DNA methodology.The PCR products are purified, restriction digested, and inserted intothe unique NdeI and XhoI or SacII sites of vector pAMG21 (ATCC accessionno. 98113) and transformed into the prototrophic E. coli 393 or 2596.Other commonly used E. coli expression vectors and host cells are alsosuitable for expression. After transformation, the clones are selected,plasmid DNA is isolated and the sequence of the OPG binding proteininsert is confirmed.

pAMG21-Murine OPG Binding Protein [75-3161

This construct was engineered to be 242 amino acids in length and havethe following N-terminal and C-terminal residues,NH₂-Met(75)-Asp-Pro-Asn-Arg-Gln-Asp-Ile-Asp(316)-COOH. The template tobe used for PCR was pcDNA/32D-F3 and oligonucleotides #1581-72 and#1581-76 were the primer pair to be used for PCR and cloning this geneconstruct.

1581-72: (SEQ ID NO:_(——))5′-GTTCTCCTCATATGGATCCAAACCGTATTTCTGAAGACAGCACTCAC TGCTT-3′

1581-76: (SEQ ID NO:_(——)) 5′-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3′pAMG21-Murine OPG Binding Protein [95-3161

This construct was engineered to be 223 amino acids in length and havethe following N-terminal and C-terminal residues,NH₂-Met-His(95)-Glu-Asn-Ala-Gly-Gln-Asp-Ile-Asp(316)-COOH. The templateused for PCR was pcDNA/32D-F3 and oligonucleotides #1591-90 and #1591-95were the primer pair used for PCR and cloning this gene construct.

1591-90: (SEQ ID NO:_(——))5′-ATTTGATTCTAGAAGGAGGAATAACATATGCATGAAAACGCAGGTCT GCAG-3′

1591-95: (SEQ ID NO:_(——))5′-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3′pAMG21-Murine OPG Binding Protein [107-3161]

This construct was engineered to be 211 amino acids in length and havethe following N-terminal and C-terminal residues,NH₂-Met-Ser(107)-Glu-Asp-Thr-Leu-Gln-Asp-Ile-Asp(316)-COOH. The templateused for PCR was pcDNA/32D-F3 and oligonucleotides #1591-93 and #1591-95were the primer pair used for PCR and cloning this gene construct.

1591-93: (SEQ ID NO:_(——))5′-ATTTGATTCTAGAAGGAGGAATAACATATGTCTGAAGACACTCTGCC GGACTCC-3′

1591-95: (SEQ ID NO:_(——))5′-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3′pAMG21-Murine OPG Binding Protein [118-3161]

This construct was engineered to be 199 amino acids in length and havethe following N-terminal and C-terminal residues,NH₂-Met(118)-Lys-Gln-Ala-Phe-Gln-Gln-Asp-Ile-Asp(316)-COOH. The templateused for PCR was pcDNA/32D-F3 and oligonucleotides #1591-94 and #1591-95were the primer pair used for PCR and cloning this gene construct.

1591-94: (SEQ ID NO:_(——))5′-ATTTGATTCTAGAAGGAGGAATAACATATGAAACAAGCTTTTCAGGG G-3′

1591-95: (SEQ ID NO:_(——))5′-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3′pAMG21-Murine OPG Binding Protein [128-3161]

This construct was engineered to be 190 amino acids in length and havethe following N-terminal and C-terminal residues,NH₂-Met-Lys(128)-Glu-Leu-Gln-His-Gln-Asp-Ile-Asp(316)-COOH. The templateused for PCR was pcDNA/32D-F3 and oligonucleotides #1591-91 and #1591-95were the primer pair used for PCR and cloning this gene construct.

1591-91: (SEQ ID NO:_(——))5′-ATTTGATTCTAGAAGGAGGAATAACATATGAAAGAACTGCAGCACAT TGTG-3′

1591-95: (SEQ ID NO:_(——))5′-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3′pAMG21-Murine OPG Binding Protein [137-3161]

This construct was engineered to be 181 amino acids in length and havethe following N-terminal and C-terminal residues,NH₂-Met-Gln(137)-Arg-Phe-Ser-Gly-Gln-Asp-Ile-Asp(316)-COOH. The templateused for PCR was pcDNA/32D-F3 and oligonucleotides #1591-92 and #1591-95were the primer pair used for PCR and cloning this gene construct.

1591-92: (SEQ ID NO:_(——))5′-ATTTGATTCTAGAAGGAGGAATAACATATGCAGCGTTTCTCTGGTGC TCCA-3′

1591-95: (SEQ ID NO:_(——))5′-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3′pAMG21-Murine OPG Binding Protein [146-3161]

This construct is engineered to be 171 amino acids in length and havethe following N-terminal and C-terminal residues,NH₂-Met(146)-Glu-Gly-Ser-Trp-Gln-Asp-Ile-Asp(316)-COOH. The template tobe used for PCR is pAMG21-murine OPG binding protein [75-316] describedabove and oligonucleotides #1600-98 and #1581-76 will be the primer pairto be used for PCR and cloning this gene construct.

1600-98: (SEQ ID NO:_(——))5′-GTTCTCCTCATATGGAAGGTTCTTGGTTGGATGTGGCCCA-3′

1581-76: (SEQ ID NO:_(——)) 5′-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3′pAMG21-Murine OPG Binding Protein [156-3161]

This construct is engineered to be 162 amino acids in length and havethe following N-terminal and C-terminal residues,NH₂-Met-Arg(156)-Gly-Lys-Pro-Gln-Asp-Ile-Asp(316)-COOH. The template tobe used for PCR is pAMG21-murine OPG binding protein [158-316] below andoligonucleotides #1619-86 and #1581-76 will be the primer pair to beused for PCR and cloning this gene construct.

1619-86: (SEQ ID NO:_(——))5′-GTTCTCCTCATATGCGTGGTAAACCTGAAGCTCAACCATTTGCA-3′

1581-76: (SEQ ID NO:_(——)) 5′-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3′pAMG21-Murine OPG binding protein [158-3161]

This construct was engineered to be 160 amino acids in length and havethe following N-terminal and C-terminal residues,NH₂-Met-Lys(158)-Pro-Glu-Ala-Gln-Asp-Ile-Asp(316)-COOH. The template tobe used for PCR was pcDNA/32D-F3 and oligonucleotides #1581-73 and#1581-76 were the primer pair to be used for PCR and cloning this geneconstruct.

1581-73: (SEQ ID NO:_(——))5′-GTTCTCCTCATATGAAACCTGAAGCTCAACCATTTGCACACCTCACC ATCAAT-3′

1581-76: (SEQ ID NO:_(——)) 5′-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3′pAMG21-Murine OPG Binding Protein [166-3161]

This construct is engineered to be 152 amino acids in length and havethe following N-terminal and C-terminal residues,NH₂-Met-His(166)-Leu-Thr-Ile-Gln-Asp-Ile-Asp(316)-COOH. The template tobe used for PCR is pcDNA/32D-F3 and oligonucleotides #1581-75 and#1581-76 will be the primer pair to be used for PCR and cloning thisgene construct.

1581-75: (SEQ ID NO:_(——))5′-GTTCTCCTCATATGCATTTAACTATTAACGCTGCATCTATCCCATCG GGTTCCCATAAAGTCACT-3′

1581-76: (SEQ ID NO:_(——)) 5′-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3′pAMG21-Murine OPG Binding Protein [168-3161]

This construct is engineered to be 150 amino acids in length and havethe following N-terminal and C-terminal residues,NH₂-Met-Thr(168)-Ile-Asn-Ala-Gln-Asp-Ile-Asp(316)-COOH. The template tobe used for PCR is pcDNA/32D-F3 and oligonucleotides #1581-74 and#1581-76 will be the primer pair to be used for PCR and cloning.

1581-74: (SEQ ID NO:_(——))5′-GTTCTCCTCATATGACTATTAACGCTGCATCTATCCCATCGGGTTCC CATAAAGTCACT-3′

1581-76: (SEQ ID NO:_(——)) 5′-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3′It is understood that the above constructs are examples and one skilledin the art may readily obtain other forms of OPG binding protein usingthe general methodology presented her.

Recombinant bacterial constructs pAMG21-murine OPG binding protein[75-316], [95-316], [107-316], [118-316], [128-316], [137-316], and[158-316] have been cloned, DNA sequence confirmed, and levels ofrecombinant gene product expression following induction has beenexamined. All constructs produced levels of recombinant gene productwhich was readily visible following SDS polyacrylamide gelelectrophoresis and coomassie staining of crude lysates. Growth oftransformed E. coli 393 or 2596, induction of OPG binding proteinexpression and isolation of inclusion bodies containing OPG bindingprotein is done according to procedures described in U.S. Ser. No.08/577,788 filed Dec. 22, 1995. Purification of OPG binding proteinsfrom inclusion bodies requires solubilization and renaturing of OPGbinding protein using procedures available to one skilled in the art.Recombinant murine OPG binding protein [158-316] was found to beproduced mostly insolubly, but about 40% was found in the solublefraction. Recombinant protein was purified from the soluble fraction asdescribed below and its bioactivity examined.

EXAMPLE 7 Purification of Recombinant Murine OPG Ligand [158-316]

Frozen bacterial cells harboring expressed murine OPG binding protein(158-316) were thawed and resuspended in 20 mM tris-HCl pH 7.0, 10 mMEDTA. The cell suspension (20% w/v) was then homogenized by three passesthrough a microfluidizer. The lysed cell suspension was centrifuged in aJA14 rotor at 10,000 rpm for 45 minutes. SDS-PAGE analysis showed a bandof approximately 18 kd molecular weight present in both inclusion bodiesand the supernatant. The soluble fraction was then applied to aPharmacia SP Sepharose 4FF column equilibrated with 10 mM MES pH 6.0.The OPG binding protein was eluted with a 20 column volume gradient of0-0.4M NaCl in MES pH 6.0. Fractions containing OPG binding protein werethen applied to an ABX Bakerbond column equilibrated with 20 mM MES pH6.0. OPG binding protein was eluted with a 15 CV gradient of 0-0.5M NaClin MES pH 6.0. The final product was over 95% homogeneous by SDS-PAGE.N-terminal sequencing gave the following sequence:Met-Lys-Pro-Glu-Ala-Gln-Pro-Phe-Ala-His which was identified to thatpredicted for a polypeptide starting at residue 158 (with an initiatormethionine). The relative molecular weight of the protein duringSDS-PAGE does not change upon reduction.

EXAMPLE 8 In vitro Bioactivity of Recombinant Soluble OPG-bp

Recombinant OPG protein has previously been shown to block vitaminD3-dependent osteoclast formation from bone marrow and spleen precursorsin an osteoclast forming assay as described in U.S. Ser. No. 08/577,788.Since OPG binding protein binds to OPG, and is a novel member of the TNFfamily of ligands, it is a potential target of OPG bioactivity.Recombinant soluble OPG binding protein (158-316), representing theminimal core TNFα-like domain, was tested for its ability to modulateosteoclast differentiation from osteoclast precursors. Bone marrow cellswere isolated from adult mouse femurs, and treated with M-CSF. Thenon-adherent fraction was co-cultured with ST2 cells in the presence andabsence of both vitamin D3 and dexamethasone. As previously shown,osteoclasts develop only from co-cultures containing stromal cells(ST2), vitamin D3 and dexamethasone. Recombinant soluble OPG bindingprotein was added at varying concentrations ranging from 0.16 to 500ng/ml and osteoclast maturation was determined by TRAP solution assayand by visual observation. OPG binding protein strongly stimulatedosteoclast differentiation and maturation in a dose dependent manner,with half-maximal effects in the 1-2 ng/ml range, suggesting that itacts as an potent inducer of osteoclastogenesis in vitro (FIG. 5). Theeffect of OPG binding protein is blocked by recombinant OPG (FIG. 6).

To test whether OPG binding protein could replace the stroma and addedsteroids, cultures were established using M-CSF at varyingconcentrations to promote the growth of osteoclast precursors andvarious amounts of OPG binding protein were also added. As shown in FIG.6, OPG binding protein dose dependently stimultated TRAP activity, andthe magnitude of the stimulation was dependent on the level of addedM-CSF suggesting that these two factors together are pivotal forosteoclast development. To confirm the biological relevance of this lastobservation, cultures were established on bovine cortical bone slicesand the effects of M-CSF and OPG binding protein either alone ortogether were tested. As shown in FIG. 7, OPG binding protein in thepresence of M-CSF stimulated the formation of large TRAP positiveosteoclasts that eroded the bone surface resulting in pits. Thus, OPGbinding protein acts as an osteoclastogenesis stimulating(differentiation) factor. This suggests that OPG blocks osteoclastdevelopment by sequestering OPG binding protein.

EXAMPLE 9

In vivo Activity of Recombinant Soluble OPG Binding Protein

Based on in vitro studies, recombinant murine OPG binding protein[158-316] produced in E.coli is a potent inducer of osteoclastdevelopment from myeloid precursors. To determine its effects in vivo,male BDF1 mice aged 4-5 weeks (Charles River Laboratories) receivedsubcutaneous injections of OPG binding protein [158-316] twice a day forthree days and on the morning of the fourth day (days 0, 1, 2, and 3).Five groups of mice (n=4) received carrier alone, or 1, 5, 25 or 100μg/of of OPG binding protein [158-316] per day. An additional 5 groupsof mice (n=4) received the above doses of carrier or of OPG bindingprotein [158-316] and in addition received human Fc-OPG [22-194] at 1mg/Kg/day (approximately 20 μg/day) by single daily subcutaneousinjection. Whole blood ionized calcium was determined prior to treatmenton day 0 and 3-4 hours after the first daily injection of of OPG bindingprotein [158-316] on days 1, 2, and 3. Four hours after the lastinjection on day 3 the mice were sacrificed and radiographs were taken.

Recombinant of OPG binding protein [158-316] produced a significantincrease in blood ionized calcium after two days of treament at dose of5 μg/day and higher (FIG. 8). The severity of the hypercalcemiaindicates a potent induction of osteoclast activity resulting fromincreased bone resorption. Concurrent OPG administration limitedhypercalcemia at doses of OPG binding protein [158-316] of 5 and 25μg/day, but not at 100 μg/day. These same animal were analysed byradiaography to determine if there were any effects on bone mineraldensity visible by X-ray (FIG. 9). Recombinant of OPG binding protein[158-316] injected for 3 days decreased bone density in the proximaltibia of mice in a dose-dependent manner. The reduction in bone densitywas particularly evident in mice receiving 100 μg/d confirming that theprofound hypercalcemia in these animals was produced from increased boneresorption and the resulting release of calcium from the skeleton. Thesedata clearly indicate that of OPG binding protein [158-316] acts in vivoto promote bone resorption, leading to systemic hypercalcemia, andrecombinant OPG abbrogates these effects.

EXAMPLE 10 Cloning and Expression of Soluble OPG Binding Protein inMammalian Cells

The full length clone of murine and human OPG binding protein can beexpressed in mammalian cells as previously described in Example 2.Alternatively, the cDNA clones can be modified to encode secreted formsof the protein when expressed in mammalian cells. To do this, thenatural 5′ end of the cDNA encoding the intiation codon, and extendingapproximately through the first 69 amino acid of the protein, inludingthe transmembrane spanning region, could be replaced with a signalpeptide leader sequence. For example, DNA sequences encoding theinitiation codon and signal peptide of a known gene can be spliced tothe OPG binding protein cDNA sequence beginning anywhere after theregion encoding amino acid residue 68. The resulting recombinant clonesare predicted to produce secreted forms of OPB binding protein inmammalian cells, and should undergo post translational modificationswhich normally occur in the C-terminal extracellular domain of OPGbinding protein, such as glycoslyation. Using this strategy, a secretedform of OPG binding protein was constructed which has at its 5′ end themurine OPG signal peptide, and at its 3′ end the human IgG1 Fc domain.The plasmid vector pCEP4/muOPG[22-401]-Fc as described in U.S. Ser. No.08/577,788, filed Dec. 22, 1995, was digested with NotI to cleavebetween the 3′ end of OPG and the Fc gene. The linearized DNA was thenpartially digested with XmnI to cleave only between residues 23 and 24of OPG leaving a blunt end. The restriction digests were thendephosphorylated with CIP and the vector portion of this digest(including residues 1-23 of OPG and Fc) was gel purified.

The murine OPG binding protein cDNA region encoding amino acid reisudes69-316 were PCR amplified using Pfu Polymerase (Stratagene, San Diego,Calif.) from the plasmid template using primers the followingoligonucleotides:

1602-61: CCT CTA GGC CTG TAC TTT CGA GCG CAG ATG

1602-59: CCT CTG CGG CCG CGT CTA TGT CCT GAA CTT TG

The 1602-61 oligonucleotide amplifies the 5′ end of the gene andcontains an artificial an StuI site. The 1602-59 primer amplifies the 3′end of the gene and contians an artifical NotI site. The resulting PCRproduct obtained was digested with NotI and StuI, then gel purified. Thepurified PCR product was ligated with vector, then used to transformelectrocompetent E. coli DH10B cells. The resulting clone was sequencedto confirm the intergrity of the amplified sequence and restriction sitejunctions. This plasmid was then used to transfect human 293fibroblasts, and the OPG binding protein-Fc fusion protein was collectedform culture media as previously described in U.S. Ser. No. 08/577,788,filed Dec. 22, 1995.

Using a similar strategy, an expression vector was designed that iscapable of expressing a N-terminal truncation of fused to the human IgG1Fc domain. This construct consists of the murine OPG signal peptide (aaresidue 1-21), fused in frame to murine OPG binding protein residues158-316, followed by an inframe fusion to human IgG1 Fc domain. To dothis, the plasmid vector pCEP4/murine OPG [22-401] (U.S. Ser. No.08/577,788, filed Dec. 22, 1995), was digested with HindIII and NotI toremove the entire OPG reading frame. Murine OPG binding protein,residues 158-316 were PCR amplified using from the plasmid templatepCDNA/32D-F3 using the following primers:

1616-44: CCT CTC TCG AGT GGA CAA CCC AGA AGC CTG AGG CCC AGC CAT TTG C

1602-59: CCT CTG CGG CCG CGT CTA TGT CCT GAA CTT TG

1616-44 amplifies OPG binding protein starting at residue 158 as well ascontaining residues 16-21 of the muOPG signal peptide with an artificialXhoI site.

1602-59 amplifies the 3′ end of the gene and adds an in-frame NotI site.The PCR product was digested with NotI and XhoI and then gel purified.

The Follwing complimentary primers were annealed to each other to forman adapter encoding the murine OPG signal peptide and Kozak sequencesurrounding the translation initiation site: 1616-41: AGC TTC CAC CATGAA CAA GTG GCT GTG CTG CGC ACT CCT GGT GCT CCT GGA CAT CA 1616-42: TCGATG ATG TCC AGG AGC ACC AGG AGT GCG CAG CAC AGC CAC TTG TTC ATG GTG GA

These primers were annealed, generating 5′ overhangs compatible withHindIII on the 5′ end and XhoI on the 3′ end. The digested vectorobtianed above, the annealed oligos, and the digested PCR fragment wereligated together and electroporated into DH10B cells. The resultingclone was sequenced to confirm authentic reconstruction of the junctionbetween the signal peptide, OPG binding protein fragment encodingresidues 158-316, and the IgG1 Fc domain. The recombiant plasmid waspurified, transfected into human 293 fibroblasts, and expressed as aconditioned media product as described above.

EXAMPLE 11 Peptides of the OPG Binding Protein and Preparation ofPolyclonal and Monoclonal Antibodies to the Protein

Antibodies to specific regions of the OPG binding protein may beobtained by immunization with peptides from OPG binding protein. Thesepeptides may be used alone, or conjugated forms of the peptide may beused for immunization.

The crystal structure of mature TNFα has been described [E. Y. Jones, D.I. Stuart, and N. P. C. Walker (1990) J. Cell Sci. Suppl. 13, 11-18] andthe monomer forms an antiparallel β-pleated sheet sandwich with ajellyroll topology. Ten antiparallel β-strands are observed in thiscrystal structure and form a beta sandwich with one beta sheetconsisting of strands B′BIDG and the other of strands C′CHEF [E. Y.Jones et al., ibid.] Two loops of mature TNFα have been implicated frommutagenesis studies to make contacts with receptor, these being theloops formed between beta strand B & B′ and the loop between betastrands E & F [C. R. Goh, C-S. Loh, and A. G. Porter (1991) ProteinEngineering 4, 785-791]. The crystal structure of the complex formedbetween TNFβ and the extracellular domain of the 55 kd TNF receptor(TNF-R55) has been solved and the receptor-ligand contacts have beendescribed [D. W. Banner, A. D'Arcy, W. Janes, R. Gentz, H-J. Schoenfeld,C. Broger, H. Loetscher, and W. Lesslauer (1993) Cell 73, 431-445]. Inagreement with mutagenesis studies described above [C. R. Goh et al.,ibid.] the corresponding loops BB′ and EF of the ligand TNFβ were foundto make the majority of contacts with the receptor in the resolvedcrystal structure of the TNFb:TNF-R55 complex. The amino acid sequenceof murine OPG binding protein was compared to the amino acid sequencesof TNFα and TNFβ. The regions of murine OPG binding proteincorresponding to the BB′ and EF loops were predicted based on thiscomparison and peptides have been designed and are described below

A. Antigen(s): Recombinant murine OPG binding protein [158-316] has beenused as an antigen (ag) for immunization of animals as described below,and serum will be examined using approaches described below. Peptides tothe putative BB′ and EF loops of murine OPG binding protein have beensynthesized and will be used for immunization; these peptides are:BB′ loop peptide: NH2--NAASIPSGSHKVTLSSWYHDRGWAKIS--COOH BB′ loop-Cyspeptide: NH2--NAASIPSGSHKVTLSSWYHDRGWAKISC--COOH EF loop peptide:NH2--VYVVKTSIKIPSSHNLM--COOH EF loop-Cys peptide:NH2--VYVVKTSIKIPSSHNLMC--COOHPeptides with a carboxy-terminal cysteine residue have been used forconjugation using approaches described in section B below, and have beenused for immunization.

B. Keyhole Limpet Hemocyanin or Bovine Serum Albumin Conjugation:Selected peptides or protein fragments may be conjugated to keyholelimpet hemocyanin (KLH) in order to increase their immunogenicity inanimals. Also, bovine serum albumin (BSA) conjugated peptides or proteinfragments may be utilized in the EIA protocol. Imject MaleimideActivated KLH or BSA (Pierce Chemical Company, Rockford, Ill.) isreconstituted in dH₂O to a final concentration of 10 mg/ml. Peptide orprotein fragments are dissolved in phosphate buffer then mixed with anequivalent mass (g/g) of KLH or BSA. The conjugation is allowed to reactfor 2 hours at room temperature (rt) with gentle stirring. The solutionis then passed over a desalting column or dialyzed against PBSovernight. The peptide conjugate is stored at −20° C. until used inimmunizations or in EIAS.

C. Immunization: Balb/c mice, (Charles Rivers Laboratories, Wilmington,Mass.) Lou rats, or New Zealand White rabbits will be subcutaneouslyinjected (SQI) with ag (50 μg, 150 μg, and 100 μg respectively)emulsified in Complete Freund's Adjuvant (CFA, 50% vol/vol; DifcoLaboratories, Detroit, Mich.). Rabbits are then boosted two or threetimes at 2 week intervals with antigen prepared in similar fashion inIncomplete Freund's Adjuvant (ICFA; Difco Laboratories, Detroit, Mich.).Mice and rats are boosted approximately every 4 weeks. Seven daysfollowing the second boost, test bleeds are performed and serum antibodytiters determined. When a titer has developed in rabbits, weeklyproduction bleeds of 50 mls are taken for 6 consecutive weeks. Mice andrats are selected for hybridoma production based on serum titer levels;animals with half-maximal titers greater than 5000 are used. Adjustmentsto this protocol may be applied by one skilled in the art; for example,various types of immunomodulators are now available and may beincorporated into this protocol.

D. Enzyme-linked Immunosorbent Assay (EIA): EIAs will be performed todetermine serum antibody (ab) titres of individual animals, and laterfor the screening of potential hybridomas. Flat bottom, high-binding,96-well microtitration EIA/RIA plates (Costar Corporation, Cambridge,Mass.) will be coated with purified recombinant protein or proteinfragment (antigen, ag) at 5 μg per ml in carbonate-bicarbonate buffer,pH 9.2 (0.015 M Na₂CO₃, 0.035 M NaHCO₃). Protein fragments may beconjugated to bovine serum albumin (BSA) if necessary. Fifty μl of agwill be added to each well. Plates will then be covered with acetatefilm (ICN Biomedicals, Inc., Costa Mesa, Calif.) and incubated at roomtemperature (rt) on a rocking platform for 2 hours or over-night at 4°C. Plates will be blocked for 30 minutes at rt with 250 μl per well 5%BSA solution prepared by mixing 1 part BSA diluent/blocking solutionconcentrate (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.)with 1 part deionized water (dH₂O). Blocking solution having beendiscarded, 50 μl of serum 2-fold dilutions (1:100 through 1:12, 800) orhybridoma tissue culture supernatants will be added to each well. Serumdiluent is 1% BSA (10% BSA diluent/blocking solution concentrate diluted1:10 in Dulbecco's Phosphate Buffered Saline, D-PBS; Gibco BRL, GrandIsland, N.Y.)) while hybridoma supernatants are tested undiluted. In thecase of hybridoma screening, one well is maintained as a conjugatecontrol, and a second well as a positive ab control. Plates are againincubated at rt, rocking for 1 hour, then washed 4 times using a 1×preparation of wash solution 20× concentrate (Kirkegaard and PerryLaboratories, Inc., Gaithersburg, Md.) in dH₂O. Horseradish peroxidaseconjugated secondary ab (Boeringer Mannheim Biochemicals, Indianapolis,Ind.) diluted in 1% BSA is then incubated in each well for 30 minutes.Plates are washed as before, blotted dry, and ABTS peroxidase singlecomponent substrate (Kirkegaard and Perry Laboratories, Inc.,Gaithersburg, Md.) is added. Absorbance is read at 405 nm for each wellusing a Microplate EL310 reader. (Bio-tek Instruments, Inc., Winooski,Vt.). Half-maximal titre of serum antibody is calculated by plotting thelog₁₀ of the serum dilution versus the optical density at 405, thenextrapolating at the 50% point of the maximal optical density obtainedby that serum. Hybridomas are selected as positive if optical densityscores greater than 5-fold above background. Adjustments to thisprotocol may be applied; in example, conjugated secondary antibody maybe chosen for specificity or non-cross-reactivity.

E. Cell Fusion: The animal selected for hybridoma production isintravenously injected with 50 to 100 μg of ag in PBS. Four days later,the animal is sacrificed by carbon dioxide and its spleen collectedunder sterile conditions into 35 ml Dulbeccos' Modified Eagle's Mediumcontaining 200 U/ml Penicillin G, 200 μg/ml Streptomycin Sulfate, and 4mM glutamine (2× P/S/G DMEM). The spleen is trimmed of excess fattytissue, then rinsed through 4 dishes of clean 2× P/S/G DMEM. It is nexttransferred to a sterile stomacher bag (Tekmar, Cincinnati, Ohio)containing 10 ml of 2× P/S/G DMEM and disrupted to single cellsuspension with the Stomacher Lab Blender 80 (Seward Laboratory UACHouse; London, England). As cells are released from the spleen capsuleinto the media, they are removed from the bag and transferred to asterile 50 ml conical centrifuge tube (Becton Dickinson and Company,Lincoln Park, N.J.). Fresh media is added to the bag and the process iscontinued until the entire cell content of the spleen is released. Thesesplenocytes are washed 3 times by centrifugation at 225×g for 10minutes.

Concurrently, log phase cultures of myeloma cells, Sp2/0-Ag14 orY3-Ag1.2.3 for mouse or rat splenocyte fusions, respectively, (AmericanType Culture Collection; Rockville, Md.) grown in complete medium (DMEM,10% inactivated fetal bovine serum, 2 mM glutamine, 0.1 mM non-essentialamino acids, 1 mM sodium pyruvate, and 10 mM hepes buffer; GibcoLaboratories, Grand Island, N.Y.) are washed in similar fashion. Thesplenocytes are combined with the myeloma cells and pelleted once again.The media is aspirated from the cell pellet and 2 ml of polyethyleneglycol 1500 (PEG 1500; Boehringer Mannheim Biochemicals, Indianapolis,Ind.) is gently mixed into the cells over the course of 1 minute.Thereafter, an equal volume of 2× P/S/G DMEM is slowly added. The cellsare allowed to fuse at 37° C. for 2 minutes, then an additional 6 ml of2× P/S/G DMEM is added. The cells are again set at 37° C. for 3 minutes.Finally, 35 ml of 2× P/S/G DMEM is added to the cell suspension, and thecells pelleted by centrifugation. Media is aspirated from the pellet andthe cells gently resuspended in complete medium. The cells aredistributed over 96-well flat-bottom tissue culture plates (BectonDickinson Labware; Lincoln Park, N.J.) by single drops from a 5 mlpipette. Plates are incubated overnight in humidified conditions at 37°C. 5% CO₂. The next day, an equal volume of selection medium is added toeach well. Selection consists of 0.1 mM hypoxanthine, 4×10⁻⁴ mMaminopterin, and 1.6×10⁻² mM thymidine in complete medium. The fusionplates are incubated for 7 days followed by 2 changes of medium duringthe next 3 days; HAT selection medium is used after each fluid change.Tissue culture supernatants are taken 3 to 4 days after the last fluidchange from each hybrid-containing well and tested by EIA for specificantibody reactivity. This protocol has been modified by that in Hudsonand Hay, “Practical Immunology, Second Edition”, Blackwell ScientificPublications.

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

1. An isolated nucleic acid encoding an osteoprotegerin binding proteinselected from the group consisting of: a) the nucleic acid sequence asin FIG. 1 (SEQ ID NO:______) and FIG. 4 (SEQ ID NO:______); b) nucleicacids which hybridize to the polypeptide coding regions as shown in FIG.1 (SEQ ID NO:______) and FIG. 4 (SEQ ID NO:______) and remain hybridizedunder high stringency conditions; and c) nucleic acids which aredegenerate to the nucleic acids of (a) or (b).
 2. The nucleic acid ofclaim 1 which is cDNA, genomic DNA, synthetic DNA or RNA.
 3. Apolypeptide encoded by the nucleic acid of claim
 1. 4. The nucleic acidof claim 1 including one or more codons preferred for Escherichia coliexpression.
 5. The nucleic acid of claim 1 having a detectable labelattached thereto.
 6. A nucleic acid encoding a polypeptide comprisingthe amino acid sequence of residues 1-316 and residues 70-316 as shownin FIG. 1 (SEQ ID NO:______).
 7. A nucleic acid encoding a polypeptidecomprising amino acid sequence of residues 1-317 and residues 69-317 asshown in FIG. 4 (SEQ ID NO:______) ;
 8. A nucleic acid encoding asoluble osteoprotegerin binding protein.
 9. The nucleic acid of claim 8encoding a polypeptide comprising residues 69-317 as shown in FIG. 4(SEQ ID NO:______) and truncations thereof;
 10. An expression vectorcomprising the nucleic acid of claims 1 and
 9. 11. The expression vectorof claim 10 wherein the nucleic acid comprises the polypeptide-encodingregion as shown in FIG. 1 (SEQ ID NO:______) and FIG. 4 (SEQ IDNO:______);
 12. A host cell transformed or transfected with theexpression vector of claim
 10. 13. The host cell of claim 12 which is aeucaryotic or procaryotic cell.
 14. The host cell of claim 13 which isEscherichia coli.
 15. A process for the production of an osteoprotegerinbinding protein comprising: growing under suitable nutrient conditionshost cells transformed or transfected with the nucleic acid of claim 1;and isolating the polypeptide product of the expression of the nucleicacid.
 16. A polypeptide produced by the process of claim
 15. 17. Apurified and isolated osteoprotegerin binding protein, or fragment,analog, or derivative thereof.
 18. The protein of claim 17 which is ahuman osteoprotegerin.
 19. The protein of claim 17 having the amino acidsequence as shown in FIG. 1 (SEQ ID NO:______) and FIG. 4 (SEQ IDNO:______).
 20. The protein of claim 17 which has been covalentlymodified with a water-soluble polymer.
 21. The protein of claim 20wherein the polymer is polyethylene glycol.
 22. The protein of claim 17which is a soluble osteoprotegerin binding protein.
 23. The protein ofclaim 22 comprising the amino acid sequence from residues 70-316inclusive as shown in FIG. 1 (SEQ ID NO: ______), or a fragment, analog,or derivative thereof.
 24. The protein of claim 22 comprising the aminoacid sequence from residues 69-317 inclusive as shown in FIG. 4 (SEQ IDNO:______) and truncations thereof.
 25. An antibody or fragment thereofwhich specifically binds an osteoprotegerin binding protein.
 26. Theantibody of claim 25 which is a monoclonal antibody.
 27. A method fordetecting the presence of an osteoprotegerin binding protein in abiological sample comprising: incubating the sample with the antibody ofclaim 25 under conditions that allow binding of the antibody to theosteoprotegerin binding protein; and detecting the bound antibody.
 28. Amethod for detecting the presence of osteoprotegerin in a biologicalsample comprising: incubating the sample with an osteoprotegerin bindingprotein under conditions that allow binding of the protein toosteoprotegerin; and measuring the bound osteoprotegerin bindingprotein.
 29. A method to assess the ability of a candidate compound tobind to an osteoprotegerin binding protein comprising: incubating theosteoprotegerin binding protein with the candidate compound underconditions that allow binding; and measuring the bound compound.
 30. Themethod of claim 29 wherein the compound is an agonist or an antagonistof an osteoprotegerin binding protein.
 31. A method of regulatingexpression of an osteoprotegerin binding protein in an animal comprisingadministering to the animal a nucleic acid complementary to the nucleicacids as shown in FIG. 1 (SEQ ID NO:______) and FIG. 4 (SEQ ID NO:______).
 32. A pharmaceutical composition comprising a therapeuticallyeffective amount of an osteoprotegerin binding protein in apharmaceutically acceptable carrier, adjuvant, solubilizer, stabilizerand/or anti-oxidant.
 33. The composition of claim 32 wherein theosteoprotegerin binding protein is a human osteoprotegerin bindingprotein.
 34. A method of treating bone disease in a mammal comprisingadministering a therapeutically effective amount of a modulator of anosteoprotegerin binding protein.
 35. The method of claim 34 wherein themodulator is a soluble form of an osteoprotegerin binding protein. 36.The method of claim 35 wherein the modulator is an antibody, or fragmentthereof, which specifically binds an osteoprotegerin binding protein.